Chromatography antigen-antibody interactions

Immunoaffinity column chromatography Antigen-antibody interaction a b [25]... 

Immunoaffinity chromatography utilizes the high specificity of antigen—antibody interactions to achieve a separation. The procedure typically involves the binding, to a soHd phase, of a mouse monoclonal antibody which reacts either directly with the protein to be purified or with a closely associated protein which itself binds the product protein. The former approach has been appHed in the preparation of Factor VIII (43) and Factor IX (61) concentrates. The latter method has been used in the preparation of Factor VIII (42) by immobilization of a monoclonal antibody to von WiHebrand factor [109319-16-6] (62), a protein to which Factor VIII binds noncovalenfly. Further purification is necessary downstream of the immunoaffinity step to remove... 

Affinity chromatography (12) has become an important tool in the isolation of purified fractions of such substances as enzymes. Advantage is taken of specific interactions such as antigen-antibody interactions. One substance of the pair (e.g. antigen) is bonded to a support. When a mixture is passed through the column, the specific interaction retains the corresponding antibody relative to other substances. A change of mobile phase conditions then elutes the pure antibody. This method has a real potential for analysis of specific proteins in body fluids. 

Photoswitchable sub strate-protein / cofactor- enzyme/antigen-antibody interactions Light-stimulated affinity chromatography Immobilization of photoactive separation component on solid matrices... 

Since monoclonal antibodies recognize a particular epitope on the antigen, they tend to be less cross-reactive than polyclonal antibodies in Western blotting and ELISAs. Also, because the interaction takes place with a defined affinity (dissociation constant), monoclonal antibodies are frequently preferred in the technique of im-munoaffinity chromatography (see Introduction to Experiment 2). The wide variety of antigen-antibody interactions that are present in a polyclonal antibody preparation makes it difficult to define a mobile-phase buffer that will promote the elution of the antigen of interest in good yield from an im-munoaffinity column prepared with polyclonal antibodies. 

In affinity chromatography the unique and specific biological interaction of the analyte and ligand is used for the separation (Figure 6-4). The specificity resulting from enzyme-substrate, hormone-receptor, or antigen-antibody interactions has been used in this type of chromatography. 

Now that you understand the basis for the interactions between functional groups in water, you also understand the basis for most interactions DNA-DNA, DNA-RNA, DNA-protein, RNA-protein, protein-protein, protein-ligand, enzyme-substrate (Get the picture ), antibody-antigen, protein-chromatography column—it s all the same stuff. 

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